ABSTRACT It is now becoming increasingly clear that microglia play a key role in the development of neurodegenerative diseases (ND) such as Alzheimer?s disease (AD). Microglia are highly plastic and respond to environmental cues by switching to a plethora of activation states, some of which are adaptive, while others are maladaptive. Understanding the heterogeneity of microglial activation in the context of AD may help design effective therapeutics that dampen the detrimental?but augment the beneficial?effects of activated microglia. In recent years, single-cell RNA seq (scRNAseq) analyses of microglia ex vivo have helped to define microglial heterogeneity in unprecedented detail. A striking feature of these studies is the identification of microglial clusters with common transcriptional profiles that arise during development and disease, characterized by (in part) altered expression of genes involved in lipid and lipoprotein metabolism. This is consistent with our current understanding of macrophage metabolism, where homeostatic macrophages maintain high rates of mitochondrial oxidative phosphorylation and fatty acid oxidation, compared to LPS-stimulated macrophages that metabolically shift towards glycolysis and fatty acid synthesis. In support, our preliminary data suggest that microglia show a similar metabolic polarization in vitro. Since microglial metabolism is one of the most prominent and consistent changes that follow activation/polarization, we hypothesize that microglial metabolism is a key component of the phenotypic switching involved in the development of AD. Moreover, we hypothesize that modulation of microglial metabolism may be a novel strategy to prevent, delay or reverse the onset of AD. However, our understanding of microglial metabolism is largely extrapolated from peripheral macrophages. In addition, scRNAseq studies may not be representative since the process of microglial isolation can profoundly alter cellular metabolism. In fact, no measurements of endogenous microglial metabolism are currently available. Fluorescence lifetime imaging microscopy (FLIM), is a state-of-the-art technique that measures the endogenous fluorescence of metabolic co-factors (e.g NADH and FAD), which have altered lifetimes depending on the metabolic status of the cell. In the proposed study, we will use FLIM as an innovative methodology to make precise measurements of microglial metabolism in situ. We will use FLIM to measure endogenous microglial metabolism in the early and aged brain to determine the metabolism of microglia in their native environment, and throughout life (AIM I). We will also perform FLIM in 5XFAD mice to define microglial metabolism in situ during AD pathogenesis (AIM II). This study will not only help develop a novel tool with which to measure microglial metabolism, but will also be the first the determine microglial metabolism in situ, guiding future studies of metabolic interventions to treat AD and beyond.